KR101290803B1 - Method for freezing, thawing and transferring of mammalian fertilized embryo using single general straw - Google Patents

Abstract

PURPOSE: A method for freezing, thawing, and transferring of mammalian fertilized egg using single general straw is provided to combine a series of freezing, thawing, and transferring of mammalian fertilized egg and perform the same. CONSTITUTION: A method for freezing, thawing, and transferring of mammalian fertilized egg comprises the following steps: (a) The mammalian fertilized egg except for human is pre-processed to freeze. (b) As a step for loading the pre-processed mammalian fertilized egg and thawing solution onto the straw, the loading is performed in the following order. a terminal of the straw - a cotton wick - air layer - thawing solution - freezing solution including the fertilized egg - the other terminal of the straw (c) The straw loaded the mammal fertilized egg is dipped in the liquid nitrogen and frozen. (d) The frozen straw is separated from the liquid nitrogen and thawed using a body temperature. (e) The mammal fertilized egg is transferred using the thawed straw.

Description

Method for Freezing, Thawing and Transferring of Mammalian Fertilized Embryo Using Single General Straw}

The present invention relates to a method of freezing, thawing and transplanting a mammalian fertilized egg using a straw. More specifically, the present invention relates to a method of freezing fertilized eggs of mammals other than humans by using a cell freezing straw and integrating thawing and transplantation.

In general, freezing of mammalian fertilized eggs is carried out by removing the antifreeze agent-added hypertonic solution in the steps of the first to third steps to remove moisture, dropping onto the surface of the electron copper grid and injected into the OPS In the crytop product consisting of a thin film, a freezing solution containing fertilized eggs is added dropwise to a minimum volume of microliters. In addition, the thawing of the frozen fertilized egg is carried out by immersing the freezing vessel in the thawing tank, or stepwise immersion in the hypertonic solution of a constant concentration and temperature. In addition, in order to transplant frozen and thawed fertilized eggs into surrogate mothers, the fused fertilized eggs are placed on an embryo transfer plastic straw, thereby performing a complicated process of freezing, thawing and transplanting. Additional facilities and equipment, such as advanced professional skills and sterilization environments, expensive microscopes, thermostatic baths, and various fertilized egg preservation instruments are essential. Therefore, even though a large number of excellent genotyped embryos are secured and cryopreserved, these complex freezing and thawing processes, lack of specialist personnel, and the need for expensive facilities and equipment make it difficult for farmers to transplant frozen fertilized eggs into surrogate mothers. .

Korean Patent Laid-Open Publication No. 10-2009-0123340 discloses a method of sucking a fertilized egg into a straw-type transplantation apparatus and implanting it into a fallopian tube of a surrogate mother. However, the method includes thawing a frozen fertilized egg during freezing and transplantation. It does not disclose how to use straw for the process. In addition, the Republic of Korea Patent No. 10-0874875 is equipped with fertilized eggs divided by zones such as the air layer and fertilized egg layer in the implantation straw, and in particular to suck the implantation material to help the implantation of the fertilized egg in a specific area within the straw There is disclosed a method of implanting fertilized eggs using the same, but not disclosed a freezing method using a straw.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Republic of Korea Patent Publication No. 10-2009-0123340 Republic of Korea Patent No. 10-0874875

The present inventors have made efforts to develop a method that integrates the freezing, thawing and transplantation of mammalian fertilized eggs using a single container for the simplicity, speed and cost saving of fertilizing, thawing and transplanting fertilized eggs. By dropping the fertilized egg together with thawing solution on a freezing straw in a fixed position and freezing it in liquid nitrogen, and developing the method that can be immediately used for transplantation by removing the straw from the liquid nitrogen at the time of implantation and thawing using body temperature The invention has been completed.

Accordingly, an object of the present invention is to provide a method for integrating freezing, thawing and transplanting of fertilized eggs in mammals using a straw.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a method of freezing, thawing and transplanting a mammalian fertilized egg comprising the steps of: (a) freezing pretreatment of a fertilized egg of a mammal other than a human; (b) loading the frozen pre-processed mammalian fertilized egg into the straw, wherein the straw ends-cotton cores-air layer-thawing liquid-freezing liquid containing fertilized egg-straw ends; (c) immersing and freezing the mammalian fertilized egg loaded straw in liquid nitrogen; (d) separating the frozen straw from liquid nitrogen and thawing using body temperature; And (e) transplanting the mammalian fertilized egg using thawed straw.

The present inventors have made efforts to develop a method that integrates the freezing, thawing and transplantation of mammalian fertilized eggs using a single container for the simplicity, speed and cost saving of fertilizing, thawing and transplanting fertilized eggs. By dropping the fertilized egg together with thawing solution on a freezing straw in a fixed position and freezing it in liquid nitrogen, and developing the method that can be immediately used for transplantation by removing the straw from the liquid nitrogen at the time of implantation and thawing using body temperature The invention was completed.

Hereinafter, the method of the present invention will be described in detail with each step.

Step (a): freeze pretreatment of the fertilized egg of a mammal other than human

Prior to dropping a fertilized egg from a mammal onto the straw, freeze pretreatment is first performed. The pre-freezing treatment is to reduce the shock of contact with the hypertonic fluid by removing water within the fertilized egg cells, osmotic pressure equilibrium and exposure to the hypertonic fluid prior to freezing, and damaging the fertilized egg cells caused by intracellular water freezing during freezing. To minimize this. Freezing pretreatment of the fertilized egg may be carried out using a method known in the art, for example, by exposing the fertilized egg for a predetermined time in a solution containing ethylene glycol and DMSO to remove water in the fertilized egg cell by osmotic pressure. According to a specific embodiment of the present invention, the first pretreatment and the first pretreatment of fertilized eggs exposed to a pretreatment solution containing 7.5% ethylene glycol and 7.5% DMSO further comprise 15% ethylene glycol and 15% It is carried out in two stages of the second pretreatment exposing to a solution containing DMSO.

The time of exposure to the pretreatment solution during the first pretreatment is not particularly limited, but is preferably 1-20 minutes, more preferably 3-15 minutes, even more preferably 5-10 minutes, most preferably 7 minutes. The time of exposure to the solution during the second pretreatment is preferably 10 seconds-5 minutes, more preferably 30 seconds-3 minutes, even more preferably 40 seconds-2 minutes, most preferably 1 minute. to be.

The fertilized egg of a mammal that can be applied to the method of the present invention is a fertilized egg of a mammal except a human, and examples thereof include, but are not limited to, fertilized egg of an animal such as a pig, a sheep, a dog, a cow, a goat, a horse and a mouse. It doesn't work.

Step (b): Loading the frozen pre-processed mammalian fertilized egg into straw , and dropping in the order of straw end- cotton core -air layer- thawing liquid -freezing liquid containing fertilized egg-straw end

The mammalian fertilized egg, frozen and pretreated by the above step (a), is added to the straw.

The fertilized egg dropping straw used in the present invention may use 0.25 ml freezing straw, 0.5 ml freezing straw, GMP straw or OPS straw, but most preferably, 0.25 ml freezing straw is used.

The straw is sterilized using EO (Ethylene Oxide) gas, high pressure, ultraviolet rays or gamma rays. The straw used in the present invention preferably contains a cotton yarn wick 1 inside one end, as shown in FIG.

Aspiration of the air layer 2 and the thawing solution 3 into the straw can be carried out by various methods known in the art, for example, a tuberculin syringe (1 ml) contains a cotton yarn wick in the straw. It can be attached to one end of the side and sucked air and thawing liquid.

According to one preferred embodiment of the present invention, the straw in the present invention uses a 0.25ml cell freezing straw.

Straw powder is filled in the cotton yarn wick of the straw, and thawing liquid having a capacity of about 0.5-1.5 cm in the case of 0.25ml straw using a syringe mounted at one end of the side where the cotton yarn wick is contained. It can be absorbed by straw powder filled inside cotton yarn wick. In this case, the straw powder absorbing the thawing liquid expands and solidifies firmly in the cotton yarn wick, so that the end of the straw is sealed.

The thawing solution is Phosphate buffered saline (PBS) to which 10% FBS (Fetal Bovine Serum) containing 0.1-1 M sucrose is added. The concentration of sucrose in the thawing solution is more preferably 0.1-0.5 M, even more preferably 0.1-0.3 M.

Subsequently, the air layer is sucked from the cotton yarn core of the straw so that an air layer 2 having a length of 0.5 to 1.5 cm is formed, and the thawing liquid 3 is taken to occupy a length of 5-8 cm, more preferably 6-7 cm, from the air layer. Aspirate to fill.

The thawing solution is PBS (Phosphate buffered saline) added with 10% FBS and sucrose as described above, and the concentration of sucrose in the thawing solution is preferably 0.1-1 M, more preferably 0.1-0.5 M, even more. Preferably it is 0.1-0.3 M.

By dropping the thawing solution into the vicinity of the frozen egg in the straw, it is possible to quickly supply water to the fertilized egg from which moisture before freezing is removed when the frozen fertilized egg is fused, thereby facilitating recovery of the fused egg.

Finally, the freezing liquid 4 containing the fertilized egg is placed 1-4 cm away from the inlet 5 of the straw, more preferably 1-3 cm away, even more preferably 1-2 cm away. Drop it to come. In addition, the freezing liquid 4 containing the fertilized egg is dropwise added so as to be located 1-4 cm, more preferably 1-3 cm, even more preferably 1-2 cm away from the thawing solution 3.

Dropping the fertilized egg at the inlet of the straw can be performed using a Pasture pipette.

The freezing solution containing the fertilized egg may be used by suitably modifying the solution for fertilizing eggs known in the art, for example, Phosphate buffered saline containing 15% ethylene glycol, 7.5% DMSO and 10% FBS ) Can be used.

The amount of the drop of the fertilized egg-containing freezing liquid is not particularly limited, but is preferably 0.1-10 µl, more preferably 0.2-5 µl, even more preferably 0.3-3 µl, and most preferably 1 µl. In the present invention, since the volume of the frozen fertilizer containing the fertilized egg is very small, freezing and thawing occur rapidly, thereby minimizing damage to the fertilized egg due to freezing and thawing.

The number of fertilized eggs contained in the dropping freezing liquid may vary depending on which mammal's fertilized egg, for example, in the case of mouse fertilized egg 10 or more, in the case of bovine fertilized egg 2-3 fertilized eggs are preferably dropped.

The structure of the straw in which the thawing liquid and the fertilized egg-containing freezing liquid have been dropped by the method of the present invention is as shown schematically in FIG. 2.

Step (c): Mammalian Fertilized Egg Dropped Straw to Liquid Nitrogen Immersion to freeze

The straw, in which fertilized eggs have been loaded, is immersed in liquid nitrogen and frozen. In the method of freezing in liquid nitrogen, the straw is erected vertically, and then the cotton core portion is first contacted with liquid nitrogen, and about 50% of the straw is immersed in liquid nitrogen for about 5 seconds. The whole straw is then immersed horizontally in liquid nitrogen. The straw of the present invention includes a fertilized egg in a small amount of freezing liquid, so that the freezing of the fertilized egg can be made quickly, thereby minimizing damage to the fertilized egg due to freezing.

Step (d): separating the frozen straw from liquid nitrogen and thawing using body temperature

To use the fertilized egg for transplantation, the frozen straw is removed from the liquid nitrogen and thawed using body temperature. In the present invention, the thawing of the frozen straw is performed using body temperature without the help of a special type of water bath or apparatus, and preferably, the frozen straw is thawed by hand for a while. Since thawing of the straw does not require a special apparatus such as a water bath, the subsequent implantation of the fertilized egg can be carried out easily and quickly, and the fertilized egg transplantation can be easily completed without the help of a skilled expert. In addition, according to the method of the present invention, since the freezing liquid containing the fertilized egg is a small amount, freezing and thawing can be performed quickly, thereby minimizing damage to the fertilized egg due to freezing and thawing, thereby greatly improving the incidence of litters.

Step (e): Transplanting Mammalian Embryos Using Thawed Straw

Implantation of the fertilized egg is carried out by attaching the thawed straw to the implanter. The implanter may be one known in the art, and for example, a syringe-type implanter may be inserted into the distal end portion of the straw and transplanted into a fertilized egg.

The present invention relates to a method for integrating freezing, thawing and transplantation of mammalian fertilized eggs using a cell freezing straw. The advantages of the present invention are as follows.

(i) The method of the present invention fills the straw with thawing solution and distances the thawing solution so that the fertilized egg is loaded so that all processes of freezing, thawing and transplanting the fertilized egg are possible using a single straw.

(Ii) According to the method of the present invention, the process of freezing, thawing and transplanting the fertilized egg can be performed using a single straw, so that the cumbersome process can be simplified, thereby saving time and money.

(Iii) According to the method of the present invention, the freezing and thawing of the fertilized egg is performed within a short time, thereby reducing the damage of the fertilized egg due to freezing and thawing, thereby greatly improving the litter rate after transplantation.

(Iii) In particular, the method of the present invention allows the implantation of the fertilized egg in a simplified procedure using a straw of a very simple structure as described above, without the use of expensive microscopes or the assistance of a skilled technician at the site of fertilized egg transplantation There is an advantage that the fertilized egg can be transplanted.

1 is a view showing the structure of the straw (straw) used in the method of freezing, thawing and transplanting the fertilized egg of the present invention.
FIG. 2 is a view showing a form in which a freezing liquid including thawing solution and fertilized egg is sucked into a straw and dropped. 1: cotton yarn wick, 2: air layer, 3: thaw (melting solution), 4: freezing liquid containing fertilized eggs, 5: straw inlet.
Figure 3 is a photograph observing how the fertilized egg is dehydrated and the shape of the cell changes in accordance with the pre-freezing treatment of bovine fertilized egg.
Figure 4 shows the process of dropping and freezing the fertilized egg in the straw. Panel 1 is a process of attaching a syringe to a straw to attach a thawing liquid (melting liquid) to the straw, and is formed by soaking the cotton core with a melting liquid and sucking an air layer in the middle to fill the melting liquid. Panel 2 shows an air gap of about 0.5 cm from cotton yarn wicks and 6 cm of melt. Panel 3 is a drop of 1 μl of freezing solution containing fertilized eggs using a paste pipette in the empty space in front of the straw. Panel 4 is a photograph obtained by enlarging the freezing solution containing a fertilized egg dropped on the straw under a microscope. Panel 5 shows the completion of fertilization. Panel 6 is a photograph showing the immersion of the straw in liquid nitrogen.
Figure 5 shows the frozen straw to be taken out of the liquid nitrogen for fertilized egg implantation to melt the frozen fertilized egg using a hand.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1 Pre-freezing Treatment of Embryos

Bovine fertilized eggs were exposed to phosphate buffered saline (PBS) containing 7.5% ethylene glycol (EG), 7.5% DMSO and 10% FBS for 7 minutes, followed by 15% ethylene glycol, 15% DMSO, and 10% FBS. Exposed to PBS containing for 1 minute to remove the water of the fertilized egg. As a result of the pre-freezing treatment, the embryos were dehydrated and the cell morphology was changed, and it was confirmed that the pre-freezing treatment of the fertilized eggs was properly performed (see FIG. 3).

Example 2 Dropping and Freezing Treatment of Fertilized Eggs in Straw

A fertilized egg of a cow treated before freezing was added dropwise to a 0.25 ml freeze straw. First, a tuberculin syringe (1 ml) was attached to one end of the side containing the cotton yarn cores of the straw, and a freeze thaw (melting solution) was aspirated using a syringe and added dropwise to the straw (Fig. 4). Panel 1). Specifically, the frozen thaw solution was aspirated with a straw length of about 1 cm to wet cotton yarn wicks. Subsequently, the air was sucked so as to form an air layer about 0.5 cm away from the cotton yarn wick, and the freeze thaw solution was added dropwise in an amount of about 6 cm in length. Panel 2 of FIG. 4 shows that about 0.5 cm of the air layer is dropped from the cotton yarn wick to fill the melt with a length of about 6 cm.

Subsequently, 1 μl of the freezing liquid containing bovine fertilized eggs was added to the empty space at the inlet portion of the terminal straw opposite the cotton core (see panel 3 in FIG. 4). Panel 4 of FIG. 4 is a photograph showing an enlarged microscopic view of a freezing solution containing fertilized eggs dropped into a straw. The straw, in which the drop of fertilized eggs was completed (Panel 5 of FIG. 4), was immersed in liquid nitrogen and frozen (see Panel 6 of FIG. 4).

Example 3 Fusion of Frozen Straws

The frozen straw was taken out of liquid nitrogen for fertilized egg transplantation, and the whole straw was wrapped with hands to melt using body temperature (see FIG. 5).

Example 4 Survival and Embryo Transfer Rates of Frozen Embryos

The viability of the fertilized eggs and the production of litter after transplantation were measured by the method of the present invention. Table 1 below shows the results of measuring the survival rate and the embryo transfer rate of embryos after thawing frozen fertilized eggs according to the sucrose concentration of the thawed solution dropped on the straw.

Survival and litter production rate of frozen fertilized eggs according to the sucrose concentration of thawed solution Sucrose
density Freeze fertilized egg
Count Thawing fertilized egg
Count % Recovery Survival rate (%) Embryo transplantation rate (%) 1 M 63 63 58 (92.06) 34 (58.62) 12 (19.05) 0.5 M 59 59 56 (94.91) 21 (37.5) 14 (23.73) 0.3 M 71 71 64 (90.10) 35 (54.68) 16 (22.53) 0 M (PBS) 71 71 70 (98.59) 25 (35.7) 10 (14.08)

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (10)

Method of freezing, thawing and transplanting a mammalian fertilized egg comprising the following steps:
(a) freeze pretreatment of the fertilized egg of a mammal other than human;
(b) loading the frozen pre-processed mammalian fertilized egg and thawing solution onto the straw, in which dropping is done in the order of the straw end-cotton core-air layer-thawing solution-freezing liquid containing fertilized egg-straw end ;
(c) immersing and freezing the mammalian fertilized egg loaded straw in liquid nitrogen;
(d) separating the frozen straw from liquid nitrogen and thawing using body temperature; And
(e) transplanting the mammalian fertilized egg using thawed straw.
The method according to claim 1, wherein the freezing pretreatment of the mammalian fertilized egg in step (a) is performed by exposing the fertilized egg to PBS (Phosphate buffered saline) containing ethylene glycol and DMSO.
The method of claim 1, wherein the thawing solution in step (b) is characterized in that the phosphate buffered saline (PBS) containing 0.1-1 M sucrose.
According to claim 1, wherein the freezing solution in step (b) is characterized in that the phosphate buffered saline (PBS) containing ethylene glycol, DMSO and FBS (Fetal Bovine Serum).
The method of claim 1, wherein the straw in step (b) is a straw for freezing 0.2 ml cells.
6. The method of claim 5, wherein in step (b) the air layer in the straw is dropped to occupy a length of 0.5-1.5 cm.
6. The method according to claim 5, wherein the thawing liquid in the straw in step (b) is added dropwise to occupy 5-8 cm in length.
6. The method according to claim 5, wherein in said straw, said fertilized egg-containing freezing liquid in the straw is added dropwise at a volume of 0.1-10 mu l at a position 1-4 cm away from the thawing liquid.
The method according to claim 1, wherein the thawing using the body temperature in the step (d) is to thaw the frozen straw by hand.
2. The method of claim 1, wherein the fertilized eggs of mammals other than humans in step (a) are fertilized eggs of pigs, sheep, dogs, cattle, goats, horses, or mice.

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